Much of the information set forth herein has been published. See Barton, J. K. et al., J. Am. Chem. Soc., 1984, 106: 2172-2176 (Apr. 6, 1984); Barton, J. K. and Raphael, A., J. Am. Chem. Soc., 1984, 106: 2466-2468 (Apr. 18, 1984); Barton, J. K. et al., Proc. Natl. Acad. Sci. USA, 1984, 81:1961-1965 (Apr. 27, 1984); and Barton, J. K., J. Biom. Structure and Dynam., 1983, 1: 621-632 (Jan. 18, 1984). The above-mentioned papers were distributed by the respective publishers on the dates provided in parentheses.
Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The binding of heterocyclic compounds to DNA by intercalation, where the planar aromatic cation stacks between adjacent base pairs of the duplex (1), has been the subjected of considerable investigation (2-4).
Intercalative drugs can be strongly mutagenic, and some, as adriamycin and daunomycin, serve as potent chemotherapeutic agents (5). The small intercalators such as ethidium or proflavine in addition provide useful chemical probes of nucleic acid structure (6). Metallointercalators have been particularly useful in probing DNA structure and the intercalation process itself, because the ligands or metal may be varied in an easily controlled manner to facilitate the individual application (7).
The aromatic chromophore of the intercalative cation can provide a sensitive handle to monitor the conformation and flexibility of the helix. Many intercalators show antibacterial or anticancer activity and, because the inserted residue often resembles a base pair in shape and thickness, intercalators are commonly frame shaft mutagens. (3) Intercalation appears to require, simply, a planar heterocyclic residue, (4) and in fact cationic metal complexes which contain aromatic ligands bind to DNA by intercalation as well. (5) Platinum intercalators have uniquely provided electron dense probes for x-ray diffraction experiments. (6) Moreover the metallointercalation reagents have offered particular experimental flexibility in that both the metal and accessory ligands may be varied for individual applications. Comparisons of the binding of the intercalative 2,2'-bipyridylplatinum(II) reagent with the analogous nonintercalating bis(pyridine)platinum(II) species by fiber X-ray diffraction methods, for example, demonstrated quite simply the requirement for ligand planarity in the intercalation process (8).
The original studies of metallointercalators centered on square-planar platinum(II) complexes containing aromatic terpyridyl or phenanthroline ligands (9), and single-crystal studies of terpyridylplatinum(II) species stacked with nucleotides showed the platinum complex to insert almost fully between the base pairs (10, 11). More recently the reagent methidiumpropyl-Fe(II)EDTA, which contains a redox-active metal center tethered to an organic intercalator, has been applied in "footprinting" experiments to determine the sequence specificities of small drugs bound to DNA (12). Bis(phenanthroline)-cuprous ion (13) has similarly been employed in DNA cleavage experiments (14), and this reagent also presumably binds initially to the DNA by intercalation. (3,5,6,8-tetramethyl-1,10-phenanthroline).sub.3 Ru(II) has been reported and its use against influenza virus, fungus, yeast and leukemia investigated (98). The ability of the tris tetramethyl complex to bind to or cleave DNA has not been reported. Furthermore, enantiomers of that complex have not been separated and their respective properties compared.
Reagents of high specificity and even stereoselectivity would be desirable in the design both of potent drugs and of structural probes. For the chiral complex (phen).sub.3 Zn.sup.2+ (phen=1,10-phenanthroline) an enantiomeric preference in binding to B-DNA has been observed (15). As for the tetrahedral (phen).sub.2 Cu.sup.+ complex, and in contrast to the square-planar platinum intercalators, the octahedral coordination in the tris(phenanthroline) metal cations can permit a partial insertion of only one coordinated ligand. Thus while one ligand is stacked between base pairs, the remaining nonintercalated phenanthroline ligands should be available to direct the enantiomeric selection.
The left-handed DNA helix has received considerable attention since the original crystallographic study of the Z-DNA fragment [d(CpG)].sub.6 (16). Solution conditions that include high ionic strength (17), hydrophobic solvents (18), the presence of certain trivalent cations (19), or covalent modification with bulky alkylating agents (19-23) all facilitate the transition of a right-handed double helix into a left-handed form. This striking conformational transition was first observed for poly[d(G-C)] (17). More recently, the alternating purine-pyrimidine sequence [d(G-T)].sub.n .multidot.[d(C-A].sub.n has been shown to form Z-helices as well (24, 25). Methylation of cytosine residues at carbon-5 lends stability to Z-form DNA (19, 26) and, under physiological conditions, transitions to a left-handed structure can occur to relieve the torsional strain in underwound negatively supercoiled DNA (27-29). These latter findings suggest mechanisms for left-handed DNA formation in the cell, where such structures could be important in controlling gene expression. Negatively supercoiled simian virus 40 DNA, for example, has been found to contain potentially Z-DNA-forming alternate purine-pyrimidine regions within transcriptional enhancer sequences (30).
To explore any biological role for left-handed DNA, sensitive and selective probes are required. Assays of superhelix unwinding, NMR experiments and circular dichroism have been used in detecting Z-DNA. These methods, however, are indirect, do not assay for helix handedness, and require large quantitites of material. More recently antibodies to Z-DNA have been elicited. The antibodies provide a more sensitive means of detection. Z-DNA appears to be a strong immunogen. Anti-Z-DNA antibodies have been elicited with both brominated poly[d(G-C)] (31) and poly[d(G-C)] modified with diethylenetriamineplatinum(II) (32) as antigens. The structures of Z-DNA and in particular of a modified Z-form provide a multitude of antigenic characteristics: the left-handed helicity, the zigzag dinucleotide phosphate repeat, the protruding purine substituents in the shallow major groove. It is not surprising then that the various antibodies obtained appear specific for different localized features of Z-DNA (33). The development of a specific chemical probe, so designed as to recognize a known structural element of Z-DNA, e.g. the helix handedness, would offer a simple complementary approach but has not heretofore been reported.
Enantiomeric selectivity has been observed in the interactions of tris(phenanthroline) metal complexes with B-DNA (15, 35-35). Experiments with tris(phenanthroline)zinc(II) have indicated stereoselectivity (15); dialysis of B-DNA against the racemic mixture leads to the optical enrichment in the .LAMBDA. enantiomer. Subsequent luminescene, electrophoretic, and equilibrium dialysis studies of the well-characterized ruthenium(II) analogues have shown that the tris(phenanthroline) metal isomers bind to DNA by intercalation and it is the .DELTA. enantiomer that binds preferentially to a right-handed duplex (34, 35). The enantiomeric selectively is based on steric interactions between the nonintercalated phenanthroline ligands and the phosphate backbone. Although the right-handed propeller-like isomer intercalates with facility into a right-handed helix, steric repulsions interfere with a similar intercalation of the .LAMBDA. enantiomer.
Based on this premise, tris(phenanthroline) metal complexes appear useful in the design of probes to distinguish left-handed and right-handed DNA duplexes. The design flexibility inherent in metallointercalation reagents, in which both ligand and metal may be varied easily, makes the coordination complexes attractive probes (7, 8, 35). We have concentrated here on phenanthroline complexes of ruthenium(II) because of the high luminescence associated with their intense metal-to-ligand charge-transfer band (37, 38) and because the exchange-inert character of the low-spin d.sup.6 complexes limits racemization (20).
Furthermore, there has been considerable interest in DNA endonucleolytic cleavage reactions that are activated by metal ions, (39, 40) both for the preparation of "footprinting" reagents (41) and as models for the reactivity of some antitumor antibiotics, notably bleomycin (42) and streptonigrin. (43) The features common to these complexes are that the molecule has a high affinity for double-stranded DNA and that the molecule binds a redox-active metal ion cofactor. The delivery of high concentrations of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient DNA cleavage reaction. Additionally, cobalt(III) bleomycin cleaves DNA in the presence of light. (44)